Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
  • G01N33/533—Production of labelled immunochemicals with fluorescent label
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
  • C07D219/02—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with only hydrogen, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the ring system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
  • C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
  • C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
  • C07D219/06—Oxygen atoms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive

Definitions

• This invention relates to chemiluminescent N-alkylacridancarboxylate derivatives which allow the production of light (chemiluminescence) from the acridan by reaction with a peroxide and a peroxidase.
• This invention also relates to an improved method of generating light chemically (chemiluminescence) by the action of a peroxidase enzyme and an oxidant such as hydrogen peroxide with a group of N-alkylacridancarboxylate derivatives.
• the invention also relates to an improved method of enhancing the amount of chemiluminescence produced from this process by the use of specific substances.
• the invention also relates to the use of this method to detect the peroxidase enzyme.
• the invention also relates to the use of this method to detect hydrogen peroxide. Further, the invention relates to the use of the method to detect and quantitate various biological molecules. For example, the method may be used to detect haptens, antigens and antibodies by the technique of immunoassay, proteins by Western blotting , DNA and RNA by Southern and Northern blotting, respectively. The method may also be used to detect DNA in DNA sequencing applications. The method may additionally be used to detect enzymes which generate hydrogen peroxide such as glucose oxidase, glucose-6-phosphate dehydrogenase, galactose oxidase and the like as are generally known in the art.
• enzymes which generate hydrogen peroxide such as glucose oxidase, glucose-6-phosphate dehydrogenase, galactose oxidase and the like as are generally known in the art.
• N-Methylacridan is oxidized electrochemically to N-methylacridinium ion (P. Hapiot, J. Moiroux, J. M. Saveant, J. Am. Chem. Soc, 112(4), 1337-43 (1990); N. W. Koper, S. A. Jonker, J. W. Verhoeven, Recl. Trav.
• Oxidation of an N-alkylacridan derivative has been performed photochemically with or without a flavin compound as co-oxidant (W. R. Knappe, J. Pharm. Sci., 67(3), 318-20 (1978); G. A. Digenis, S. Shakshir, M. A. Miyamoto, H. B. Maschinenbauer, J. Pharm. Sci., 65(2), 247-51 (1976)).
• Chemiluminescence quantum yields ranged from 10 -5 to 0.1 and were found to increase as the pKa of the phenol or alcohol leaving group decreased. Quantum yields in aqueous solution were significantly lower due a competing non-luminescent decomposition of an intermediate. Addition of the cationic surfactant CTAB increased the apparent light yield 130-fold by preventing a competing dark reaction.
• Applicants' co-pending application Serial No. 08/061,810 discloses the first use of an enzyme to oxidize substituted and unsubstituted N-alkylacridancarboxylic acid derivatives to generate chemiluminescence.
• N-alkylacridancarboxylate derivatives are efficiently oxidized to produce the N-alkylacridone and blue chemiluminescence.
• acridinium esters especially when labeled to a protein or oligonucleotide suffers from two disadvantages.
• the chief problem is limited hydrolytic stability.
• Acridinium ester conjugates decompose steadily at or slightly above room
• a second disadvantage of acridinium esters is the tendency to add nucleophiles such as water at the 9-position to spontaneously form a pseudo-base intermediate which is non-luminescent and decomposes in a pH-dependent manner in a dark process.
• nucleophiles such as water at the 9-position
• pseudo-base intermediate which is non-luminescent and decomposes in a pH-dependent manner in a dark process.
• the pH of solutions containing acridinium esters must be first lowered to reverse pseudo-base formation and then raised in the presence of H 2 O 2 to produce light.
• a more fundamental limitation to the use of acridinium esters as chemiluminescent labels lies in the fact that when used as direct labels, only up to at most about 10 molecules can be attached to a protein or oligonucleotide. Coupled with the quantum efficiency for producing a photon ( ⁇ 10%), an acridinium ester-labeled analyte can generate at most one photon of light. In contrast, enzyme-labeled analytes detected by a chemiluminescent reaction can potentially generate several orders of magnitude more light per analyte molecule detected by virtue of the catalytic action of the enzyme.
• chemiluminescent compound oxidized by a peroxidase enzyme and a peroxide is a hydroxy-substituted phthalhydrazide (Akhavan-Tafti co-pending U. S. patent application No .965 , 231 , filed October 23 , 1992 ) .
• Applicant's co-pending application Serial No. 08/061,810 filed on May 17, 1993 discloses chemiluminescent N-alkylacridancarboxylic acid esters and sulfonimides which produce light upon reaction with a peroxide and a peroxidase for use in detecting peroxidase enzymes and in assays.
• Enhancers have also been employed in conjunction with the use of luminol to increase the intensity and duration of light emitted. These include benzothiazole derivatives such as D-luciferin, various phenolic compounds such as p-iodophenol and p-phenylphenol and aromatic amines (G. Thorpe, L. Kricka, in Bioluminescence and Chemiluminescence, New Perspectives, J. Scholmerich, et al, Eds., pp. 199-208 (1987)).
• benzothiazole derivatives such as D-luciferin
• various phenolic compounds such as p-iodophenol and p-phenylphenol and aromatic amines
• phenolic compounds are taken to mean hydroxylic aromatic compounds which will also include compounds such as 2-naphthol and 6-bromo-2-naphthol which are known to enhance other peroxidase reactions in addition to the aforementioned substituted hydroxyphenyl compounds.
• Other compounds which function as enhancers of the chemiluminescent oxidation of amino-substituted cyclic acylhydrazides by a peroxidase include 4-(4-hydroxyphenyl)thiazole (M. Ii, H. Yoshida, Y. Aramaki, H. Masuya, T. Hada, M. Terada, M. Hatanaka, Y. Ichimori, Biochem. Biophys. Res.
• kits for conjugation of HRP with enhanced luminol chemiluminescent detection are available.
• Chemiluminescent assays using a peroxidase enzyme known in the art are not able to detect the lowest levels of certain analytes such as the thyroid hormone TSH, mainly due to the inability to detect the enzyme at extremely low levels.
• a chemiluminescent reagent which permits the detection of lower amounts of enzyme is needed for such assays.
• Enhancement of chemiluminescent reactions using polymeric and monomeric surfactants is known in the art. Enhancement may occur by affecting the outcome of one or more steps e.g. by increasing the fluorescence quantum yield of the emitter, by increasing the percentage of product molecules produced in the excited state, by increasing the fraction of molecules undergoing the chemiluminescent reaction through inhibition of competing side reactions (McCapra Accts.
• U.S. Patent No. 5,145,772 to Voyta discloses enhancement of enzymatically generated chemiluminescence from 1,2-dioxetanes in the presence of polymeric compounds. Certain cationic polymer compounds were effective chemiluminescence enhancers; nonionic polymeric compounds were generally ineffective and the lone anionic polymer, Example 45, significantly decreased light emission.
• U.S. Patent No. 4,927,769 to Chang discloses enhancement by surfactants of the chemical oxidation of acridinium esters with alkaline hydrogen peroxide. These acridinium ester compounds are discrete from compounds of the present invention in that they react without the use of enzymes. Several of the tested surfactants (see Table 2 therein) provide only marginal enhancement.
• a paper discloses enhancement by a cationic surfactant of chemiluminescence from chemical oxidation of a dialkylaminobenzofuranyl-substituted cyclic diacylhydrazide.
• An anionic surfactant was ineffective at enhancing the chemiluminescence, while a nonionic surfactant diminished light production.
• N-alkylacridancarboxylate derivatives with superior properties for use in generating chemiluminescence by the action of a peroxidase enzyme for the detection of biological materials and compounds. It is also an object of the present invention to provide an improved method and kit using N-alkylacridancarboxylate derivatives in solution or on membranes for use in generating chemiluminescence by the action of a peroxidase enzyme for the detection of peroxidase enzymes and enzyme-conjugates.
• N-alkylacridancarboxylate derivatives for use in generating chemiluminescence by the action of a peroxidase enzyme for use in nucleic acid assays in solution and on surfaces.
• N-alkylacridancarboxylate derivatives for use in generating chemiluminescence by the action of a peroxidase enzyme for detection of proteins in Western blots and DNA in Southern blots and other DNA hybridization assays.
• N-alkylacridancarboxylate derivatives for use in generating chemiluminescence by the action of a peroxidase enzyme for detection of haptens, proteins and antibodies in enzyme immunoassays.
• Figure 1 is a graph showing a comparison of the light emission profiles from a reagent containing 2',6'-difluorophenyl 10-methyl-acridan-9-carboxylate (5a) of the present invention, a reagent containing the acridan 4'-fluorophenyl 10-methylacridan-9-carboxylate and a commercial reagent containing luminol. Forty ⁇ L each of three formulations were reacted in separate experiments with 1 ⁇ L of a solution containing 1.4 x 10 -16 mol of HRP in water.
• formulations consisted of: (1) 0.05 mM acridan compound 5a in 0.01 M tris buffer, pH 8.0, 0.4 mM urea peroxide, 0.1 mM p-phenylphenol, 0.025% TWEEN 20, 1 mM EDTA; (2) an identical formulation containing 4'-fluorophenyl 10-methylacridan-9- carboxylate in place of 5a and (3) an optimized reagent containing luminol (AMERSHAM ECL, Amersham, PLC, Amersham, U.K.).
• Figure 1 shows the improved generation of light emission (in Relative Light Units, RLU) using 5a, a reagent of the present invention, under these conditions compared to the prior art compounds 4'-fluorophenyl 10-methylacridan-9-carboxylate and luminol.
• Figure 2 is a graph showing a comparison of the light emission profiles from a reagent containing 3',5'-difluorophenyl 10-methylacridan-9-carboxylate (5b) of the present invention, a reagent containing the acridan 4'-fluorophenyl 10-methylacridan-9-carboxylate and a commercial reagent containing luminol. Forty ⁇ L each of three formulations were reacted in separate experiments with 1 ⁇ L of a solution containing 1.4 ⁇ 10 -16 mol of HRP in water.
• Figure 3 is a graph showing a comparison of the light emission profiles from a reagent containing 2',4',6'-trichlorophenyl 10-methylacridan-9-carboxylate (5c) of the present invention, a reagent containing the acridan 4'-fluorophenyl 10-methylacridan-9-carboxylate and a commercial reagent containing luminol. Forty ⁇ L each of three formulations were reacted in separate experiments with 1 ⁇ L of a solution containing 1.4 ⁇ 10 -16 mol of HRP in water.
• Figure 4 is a graph showing a comparison of the light emission profiles from a reagent containing 2',4',5'-trichlorophenyl 10-methylacridan-9-carboxylate (5d) of the present invention, a reagent containing the acridan 4'-fluorophenyl 10-methylacridan-9-carboxylate and a commercial reagent containing luminol. Forty ⁇ L each of three formulations were reacted in separate experiments with 1 ⁇ L of a solution containing 1.4 ⁇ 10 -16 mol of HRP in water.
• Figure 5 is a graph showing a comparison of the light emission profiles from a reagent containing 2',3',6'-trifluorophenyl 10-methylacridan-9-carboxylate (5e) of the present invention, a reagent containing the acridan 4'-fluorophenyl 10-methylacridan-9-carboxylate and a commercial reagent containing luminol. Forty ⁇ L each of three formulations were reacted in separate experiments with 1 ⁇ L of a solution containing 1.4 ⁇ 10 -16 mol of HRP in water.
• Figure 6 is a graph showing a comparison of the light emission profiles from a reagent containing pentafluorophenyl 10-methylacridan-9-carboxylate (5f) of the present invention, a reagent containing the acridan 4'-fluorophenyl 10-methylacridan-9-carboxylate and a commercial reagent containing luminol. Forty ⁇ L each of three formulations were reacted in separate experiments with 1 ⁇ L of a solution containing 1.4 ⁇ 10 -16 mol of HRP in water.
• Figure 7 is a graph showing a comparison of the light emission profiles from a reagent containing 2',3',6'-trifluorophenyl 3-methoxy-10-methylacridan-9-carboxylate (5h) of the present invention, a reagent containing the acridan 4'-fluorophenyl 10-methylacridan-9-carboxylate and a commercial reagent containing luminol. Forty ⁇ L each of three formulations were reacted in separate experiments with 1 ⁇ L of a solution containing 1.4 ⁇ 10 -16 mol of HRP in water.
• Figure 8 is a graph showing a comparison of the linearity of detection of HRP using reagent compositions of the present invention and a commercially available optimized reagent containing luminol.
• 40 ⁇ L of a solution containing acridan 5e, 5h or a commercial reagent (AMERSHAM ECL) were mixed at room temperature with 1 ⁇ L aliquots of HRP containing the indicated amounts of enzyme.
• Light intensities from the compositions containing acridans 5e and 5h were measured at 15 min while data from the ECL reagent represent the maximum light intensity.
• S-B refers to the chemiluminescence signal (S) in RLU in the presence of HRP corrected for background chemiluminescence (B) in the absence of HRP.
• Compositions containing acridans 5e and 5h are capable of 100-fold greater sensitivity of detection than the ECL reagent.
• Figures 9A, 9B and 9C show the result of three experiments concerning Western blot analysis of human transferrin on PVDF with chemiluminescent detection using fractionated goat anti-human transferrin serum, rabbit anti-goat IgG-peroxidase conjugate.
• human transferrin loaded into the five slots was: (1) 1000 pg, (2) 200 pg, (3) 50 pg, (4) 20 pg, (5) 5 pg.
• Chemiluminescent detection was performed using: a commercial reagent (ECL) containing luminol ( Figure 9A); a reagent composition containing the acridan 4'-hydroxyphenyl 10-methylacridan-9-carboxylate previously disclosed in applicants' co-pending application Serial No.
• Figures 10A, 10B and 10C show the result of a Western blot analysis of human transferrin on nitrocellulose with chemiluminescent detection using fractionated goat anti-human transferrin serum, rabbit anti-goat IgG-peroxidase conjugate.
• Human transferrin loaded into each slot was (1) 1000 pg, (2) 200 pg, (3) 50 pg, (4) 20 pg, (5) 5 pg.
• Chemiluminescent detection was performed using: a commercial reagent (ECL) containing luminol ( Figure 10A); a reagent composition containing the acridan 4'-hydroxyphenyl 10-methylacridan-9-carboxylate previously disclosed in applicants' co-pending application Serial No. 08/061,810
• FIG 11 is a graph showing the linearity of detection of thyroid stimulating hormone (TSH) in a chemiluminescent enzyme immunoassay using a reagent of the present invention containing acridan 5h and a commercial TSH immunoassay kit.
• TSH thyroid stimulating hormone
• Figure 12 is a graph showing the application of a chemiluminescent reagent of the invention containing acridan 5h in an enzyme immunoassay of Figure 11 for human growth hormone (hGH).
• hGH human growth hormone
• the chemiluminescent assay resulted in a nonlinear calibration curve.
• S-B has the same meaning as in Figure 8.
• Figure 13 is a graph showing the linearity of detection of hGH obtained in the same chemiluminescent enzyme immunoassay by diluting the secondary antibody-HRP conjugate ten-fold.
• the chemiluminescent assay resulted in a linear calibration curve over three orders of magnitude with a lowest detected quantity of 0.05 ng/mL.
• Excellent linearity and identical analytical sensitivity resulted when light intensity was measured at either 2.5, 5, 7.5 or 10 min.
• the term S-B has the same meaning as in Figure 8.
• the calculated detection limit of the manufacturer's assay using a colorimetric endpoint is 0.05 ng/mL; the colorimetric assay generates a nonlinear calibration curve.
• Figures 14A and 14B show the result of chemiluminescent detection of a Southern blot analysis of EcoRI-restricted mouse genomic DNA on nylon using a fluorescein-labeled v-mos probe and horseradish peroxidase-anti-fluorescein conjugate.
• the reagents used for the chemiluminescent detection were: a composition of the present invention containing acridan 5e ( Figure 14A) and a commercial reagent (ECL) containing luminol ( Figure 14B).
• ECL commercial reagent
• the blots were exposed to X-OMAT AR X-ray film for 10 min after a 22 min incubation. The image has been scanned and digitally reproduced. The results show the superior image obtained with acridan 5e of the present invention.
• the present invention relates particularly to an improved acridan of the formula:
• R 1 is selected from alkyl, heteroalkyl and aralkyl groups, wherein R is any group which allows the production of light and a and b are integers between 0 and 4 and wherein Ar-O is a leaving group wherein Ar-O is selected from the group consisting of di- and polyhalosubstituted phenoxy groups which allows the production of light from the acridan by reaction with a peroxide and a peroxidase.
• Ar-O groups containing at least two halogen substituents provide unexpectedly superior performance in producing chemiluminescence and in assays.
• the present invention relates to an improvement in a reagent composition which generates light in the presence of a peroxidase which comprises:
• R 1 is selected from the group consisting of alkyl, heteroalkyl and aralkyl groups, wherein R is any group which allows the production of light and a and b are integers between 0 and 4 and wherein Ar-O is a leaving group wherein Ar-O is selected from the group consisting of di- and polyhalosubstituted phenoxy groups which allows the production of light from the acridan by reaction with a peroxide and a peroxidase;
• the present invention relates to an improved method for producing chemiluminescence which comprises reacting a peroxide compound and a peroxidase enzyme with an acridan of the formula:
• R 1 is selected from the group consisting of alkyl, heteroalkyl and aralkyl groups, wherein R is any group which allows the production of light and a and b are integers between 0 and 4 and wherein Ar-O is a leaving group wherein Ar-O is selected from the group consisting of di- and polyhalosubstituted phenoxy groups which allows the production of light from the acridan by reaction with a peroxide and a peroxidase.
• the present invention also relates to an improved method for detecting a peroxidase enzyme or an analyte linked to or capable of being linked to a peroxidase enzyme in an assay procedure by a chemiluminescent reaction, the improvement which comprises reacting an acridan with a peroxide and a peroxidase enzyme to produce light for detecting the analyte wherein the acridan is of the following formula:
• R 1 is selected from the group consisting of alkyl, heteroalkyl and aralkyl groups, wherein R is any group which allows the production of light and a and b are integers between 0 and 4 and wherein Ar-O is a leaving group wherein Ar-O is selected from the group consisting of di- and polyhalosubstituted phenoxy groups which allows the production of light from the acridan by reaction with a peroxide and a peroxidase.
• the present invention also relates to an improved method for detecting a peroxidase enzyme or an analyte linked to or capable of being linked to a peroxidase enzyme in an assay procedure by a chemiluminescent reaction, the improvement which comprises: a) providing a reagent composition which generates light in the presence of a peroxidase which comprises: an acridan of the formula:
• R 1 is selected from the group consisting of alkyl, heteroalkyl and aralkyl groups, wherein R is any group which allows the production of light and a and b are integers between 0 and 4 and wherein Ar-O is a leaving group wherein Ar-O is selected from the group consisting of di- and polyhalosubstituted phenoxy groups which allows the production of light from the acridan by reaction with a peroxide and a peroxidase; a peroxide compound which participates in the reaction of the acridan with the peroxidase; an enhancer substance which may be a phenolic compound which enhances the light production from the acridan; a chelating agent which prevents the peroxide compound from reacting prior to addition of the peroxidase to the composition; and a surfactant; and
• the present invention also relates to a kit for detecting an analyte in an assay procedure by a chemiluminescent reaction to produce light which comprises in separate containers: a) an acridan of the formula:
• R 1 is selected from the group consisting of alkyl, heteroalkyl and aralkyl groups, wherein R is any group which allows the production of light and a and b are integers between 0 and 4 and wherein Ar-O is a leaving group wherein Ar-O is selected from the group consisting of di- and polyhalosubstituted phenoxy groups which allows the production of light from the acridan by reaction with a peroxide and a peroxidase; a peroxide; optionally an enhancer substance which may be a phenolic compound which enhances the light production from the acridan; optionally a chelating agent which prevents the peroxide compound from reacting prior to addition of the peroxidase to the composition; and a surfactant; and b) a peroxidase enzyme, wherein the light is detected in the assay procedure by reacting the reagent composition with the peroxidase.
• the present invention also relates to an improved method for detecting hydrogen peroxide in an assay procedure by a chemiluminescent reaction, the improvement which comprises reacting hydrogen peroxide and a peroxidase enzyme with an acridan of the formula:
• R 1 is selected from the group consisting of alkyl, heteroalkyl and aralkyl groups, wherein R is any group which allows the production of light and a and b are integers between 0 and 4 and wherein Ar-O is a leaving group wherein Ar-O is selected from the group consisting of di- and polyhalosubstituted phenoxy groups which allows the production of light from the acridan by reaction with a peroxide and a peroxidase.
• the invention involves improved N-alkylacridancarboxylate derivatives with superior properties in one or more of the following characteristics: longer duration of light emission, higher intensity of light emission, faster rate of rise of light emission to the maximum value, lowered background chemiluminescence, improved signal/background ratio, extended storage stability of a chemiluminescent detection reagent composition, enhanced light emission on a membrane or other properties.
• the particular combinations of the groups R and Ar are chosen so as to provide a compound with one or more properties which are optimal for particular applications.
• the groups R and Ar are chosen so as to provide a compound with one or more properties which are optimal for particular applications.
• N-alkylacridancarboxylate derivatives with Ar groups bearing two or more halogen substituents are unexpectedly superior in light generating properties. It is thought that the incorporation of two or more halogen substituents in the Ar moiety produces an acridan with a better leaving group thereby accelerating and promoting the chemiluminescent reaction at the expense of competing non-chemiluminescent side reactions.
• the O-Ar group such that the pK, of the conjugate acid (Ar-OH) of the Ar-O- anion is lower than that of Ph-OH, preferably less than about 9.5.
• examples include phenols or phenolic compounds (Ar-OH) which are substituted with pK a -lowering electron withdrawing groups such as halogen atoms and hence are more ionized at a given pH.
• pK a -lowering electron withdrawing groups
• halogen atoms halogen atoms
• one or more of the groups R may be a group such as an alkyl, alkoxy or aryloxy group.
• X is a halogen atom selected from F, Cl, Br or I and m is 2 to 5.
• Another class of preferred compounds are:
• X is a halogen atom selected from F, Cl, Br or I and m is 2 to 5.
• Additional acridan compounds which have been prepared and tested for light production include phenyl 10-methylacridan-9-carboxylate; 2',6'-dimethylphenyl 10-methylacridan-9-carboxylate; 4'-fluorophenyl 10-methylacridan-9-carboxylate; 4'-iodophenyl 10-methylacridan-9-carboxylate; 4'-phenylphenyl 10-methylacridan-9-carboxylate; 3',5'-dicarbomethoxyphenyl 10-methylacridan-9-carboxylate; 4'-(N-butylaminocarbonyl)phenyl 10-methylacridan-9-carboxylate; 4'- -methoxyphenyl 10-methylacridan-9-carboxylate; 2',6'-dimethoxyphenyl 10-methylacridan-9-carboxylate; 4'-acetamidophenyl 10-methylacridan-9-carboxylate; 4'-(2-
• N-alkylacridancarboxylate derivatives of the present invention Reaction of certain N-alkylacridancarboxylate derivatives of the present invention with a peroxide and a peroxidase enzyme produces chemiluminescence with superior properties for assay applications.
• the chemiluminescence is believed to arise from the excited state of N-alklyacridone or the substituted N-alklyacridone product as shown in the generalized reaction below .
• the present invention involves a method of generating chemiluminescence from the oxidation of N-alkylacridancarboxylic acid derivatives by the action of a peroxidase enzyme, a peroxide compound and enhancers.
• the invention also relates to the use of this method to detect the peroxidase enzyme with high sensitivity.
• the invention relates to the use of the method to detect and quantitate various biological molecules which are bound to this enzyme by chemical bonds or through physical interactions.
• the invention relates to the use of the method to detect and quantitate various biological molecules which are capable of being bound to this enzyme, for example, by using a biotin-labeled analyte and streptavidin-peroxidase conjugate.
• chemiluminescence provides a direct measure of the quantity of labeled organic or biological molecule.
• the method may be used to detect haptens, antigens and antibodies by the technique of immunoassay, proteins by Western blotting, DNA and RNA by Southern and Northern blotting, respectively. The method may also be used to detect DNA in DNA sequencing applications.
• the method may additionally be used to detect hydrogen peroxide generated by enzymes such as cholesterol oxidase, glucose oxidase, glucose-6-phosphate dehydrogenase , galactose oxidase, galactose-6-phosphate dehydrogenase, and amino acid oxidase.
• enzymes such as cholesterol oxidase, glucose oxidase, glucose-6-phosphate dehydrogenase , galactose oxidase, galactose-6-phosphate dehydrogenase, and amino acid oxidase.
• the method may also therefore be used as a means to detect the enzymes mentioned above which generate hydrogen peroxide.
• the reaction of the present invention may advantageously be carried out in solution such as an aqueous buffer or on the surface of a solid support such as a bead, tube, microwell plate or a membrane.
• Suitable buffers include any of the commonly used buffers capable of maintaining a pH in the range of about 7 to about 10 for example, phosphate, borate, carbonate, tris(hydroxymethylamino)methane, glycine, tricine, 2-amino-2-methyl-1-propanol, diethanolamine and the like.
• the preferred method of practicing the invention in this regard is determined by the requirements of the particular intended use as in for example, immunoassays, Western blotting, Southern blotting etc.
• said membrane may optionally be provided in a kit.
• chemiluminescence from the oxidation of an N-alkylacridancarboxylate derivative by hydrogen peroxide and a peroxidase enzyme can be accomplished with good sensitivity. Enhancement of this reaction by incorporation of chemiluminescence-enhancing substances permits the measurement of chemiluminescence using still lower levels of the peroxidase enzyme. Coupling this enzyme to a biological molecule of interest then permits the detection of this biological molecule with great sensitivity.
• Phenolic compounds which are known to enhance other peroxidase reactions and which are found to enhance the amount of chemiluminescence in the present invention include but are not limited to: p-phenylphenol, p-iodophenol, p-bromophenol, p-hydroxycinnamic acid, 2-naphthol and
• 6-bromo-2-naphthol 6-bromo-2-naphthol.
• phenolic and aromatic amine compounds fall within the scope of this invention.
• Such compounds include firefly luciferin,
• 6-hydroxybenzothiazole 2-cyano-6-hydroxybenzothiazole, 4-(4-hydroxyphenyl)thiazole, p-chlorophenol, 2,4-dichlorophenol, 2-chloro-4-phenylphenol,
• Additives which suppress the generation of chemiluminescence from the reaction of hydrogen peroxide and N-alkylacridancarboxylate derivatives in the absence of peroxidase enzymes are employed to further improve the utility of the invention. It has also been found that certain compounds including cyclodextrins and surfactants including C 12 - C 20 alkyl sulfates and sulfonates, dextran sulfate, C 12 - C 20 alkyltrimethylammonium salts, and nonionic surfactants including polyoxyalkylene alkylphenols, polyoxyalkylene alcohols, polyoxyealkylene ethers, polyoxyalkylene sorbitol esters and the like improve the sensitivity of detection of the peroxidase enzyme in assays of the present invention by providing a better signal to background ratio. The improvement occurs through minimizing the background chemiluminescence in the absence of added peroxidase, possibly due to a slowing of the autoxidative decomposition of
• the present invention involves a solution in an aqueous buffer containing 1) a phenol enhancer or a salt of a phenol enhancer, 2) a peroxide compound wherein the peroxide compound may be hydrogen peroxide, urea peroxide, or a perborate salt, 3) an acridan compound of the invention, 4) a cation complexing agent wherein the agent may be selected from the group consisting of chelating agents such as ethylenediaminetetraacetic acid (EDTA) , diethylenetriaminepentaacetic acid (DTPA), or ethylenebis(oxyethylenenitrilo)-tetraacetic acid (EGTA) and their salts, and 5) a surfactant such as the anionic surfactant sodium dodecyl sulfate (SDS), or preferably a nonionic surfactant such as polyoxyethylenated alkylphenols, polyoxyethylenated alcohols, polyoxyethylenated ethers, poly
• a phenol compound such as p-phenylphenol
• a nonionic surfactant at a final concentration from about 5 % to 0.005 % (v/v)
• a peroxide source such as hydrogen peroxide
• This solution is contacted with the peroxidase enzyme which may either be in solution or adhered to a solid support.
• concentrations of reagents must be determined individually for each composition.
• concentration of acridan compound and enhancer in particular should be optimized with care for each case in order to produce the maximum enhancement of light emission.
• the detection reaction may be performed over a range of temperatures including at least the range 20 - 40 °C. Detection may be conveniently and advantageously carried out at ambient temperature.
• the storage life of the detection reagent composition can be significantly extended by excluding oxygen from the solution.
• Detection reagents of the present invention stored in this manner retain the ability to generate the same quantity of chemiluminescence by the action of a peroxidase enzyme for longer periods of time. Extended storage stability can result in savings in reagents and cost.
• N-alkylacridancarboxylate derivatives and compositions of the present invention containing them are increased sensitivity of detection of the peroxidase enzyme and increased stability of the N-alkylacridancarboxylate derivative to hydrolytic decomposition. Comparative experiments show a 100-fold lowering of the detection limit of HRP using a reagent composition of this invention compared to a detection reagent containing luminol and an enhancer. An additional advantage is the wider dynamic range of measurement of peroxidase concentration. An additional advantage of N-alkylacridancarboxylate derivatives is their thermal and photochemical stability and ease of purification.
• Example 1 Synthesis of Acridan Derivatives.
• Acridancarboxylate derivatives 5a-j were synthesized according to the method shown in Scheme 1 from the corresponding acridine-9-carboxylic acid.
• the groups (R). in formula (II) are all hydrogen except as otherwise specified by the substituent A
• the groups (R) b in formula (II) are all hydrogen except as otherwise specified by the substituent B.
• Example 11 Comparison of the Light Intensity-Time Profile for Detection of HRP with Compounds 5a-f, h, a Prior Art Acridan and Luminol.
• 40 ⁇ L volumes of each of three formulations were reacted with 1 ⁇ L of a solution containing 1.4 ⁇ 10 -16 mol of HRP in water.
• the formulations consisted of: (1) 0.05 mM acridan compound 5a-f or h in 0.01 M tris buffer, pH 8.0, 0.4 mM urea peroxide, 0.1 mM p-phenylphenol, 0.025% Tween 20, 1 mM EDTA; (2) an identical formulation containing the previously reported acridan 4'-fluorophenyl 10-methylacridan-9-carboxylate in place of the acridan 5a-f or h (F. McCapra, Pure Appl. Chem., 24, 611-629 (1970)) and (3) an optimized reagent containing luminol (Amersham ECL formulation and peroxide in separate bottles).
• Figures 1-7 show the improved generation of light emission using reagents containing acridans 5a-f and h of the present invention compared to the prior art reagents under these conditions.
• a matrix optimization experiment was done using acridans 5a, 5c, 5e and 5h (0.1 mM - 0.05 mM) in solutions containing p-iodophenol (0.1-2.25 mM) or p-phenylphenol (0.01-2.25 mM), urea peroxide (0.1 mM - 1 mM) , Tween 20 (0-0.6%) in tris buffer, pH 8.0 (0.01-0.2 M). Sensitivity and dynamic range were evaluated for detection of HRP in the range 1.4 ⁇ 10 -15 to 1.4 ⁇ 10 -19 mol of enzyme.
• An especially effective reagent consists of the acridan
• Example 13 Comparison of the Sensitivity of Detection of HRP with 5e, 5h or Luminol.
• the linearity of detection of HRP using reagent compositions of the present invention and a commercially available optimized reagent containing luminol (Amersham ECL) were compared. Forty ⁇ L of a solution containing acridan 5e or 5h as described in example 2 and forty ⁇ L of the commercial reagent (Amersham ECL, prepared according to the manufacturer's directions) were mixed at room temperature with 1 ⁇ L aliquots of HRP containing between 1.4 ⁇ 10 -15 and 1.4 ⁇ 10 -19 mol of enzyme.
• Figure 8 compares the linear range of HRP amount measured.
• S-B refers to the chemiluminescence signal (S) in RLU in the presence of HRP corrected for background chemiluminescence (B) in the absence of HRP.
• Reagents containing acridans 5e and 5h are capable of 100-fold greater sensitivity of detection than the ECL reagent. Measurement with the reagents containing acridans 5e or 5h could be measured at earlier times with equivalent sensitivity.
• the detection reagents are conveniently stored in two containers, the first comprising an aqueous buffer solution containing the peroxide, phenol enhancer, TWEEN 20 and EDTA, the second solution comprising the acridan compound 5 in a water-miscible organic solvent such as i:i ethanol/p-dioxane.
• acridans of the present invention is their greater stability in the final detection reagent mixture. Stability is assessed by measuring the peak light intensity from an aliquot of the solution reacted with a specified amount of HRP.
• Example 15 Stability of Horseradish Peroxidase Detection Reagent Containing 5h .
• a similar set of experiments was performed using a reagent with the same composition containing instead acridan 5h. Stability was assessed by measuring the peak light intensity from an aliquot of the solution reacted with a specified amount of HRP.
• a detection reagent incorporating acridan 5h shows superior stability compared to previous compounds and is stable for at least one day without special precautions.
• Example 16 Effect of pH of the Buffer. Detection reagent solutions according to the composition of Example 14 were prepared with either 0.01 M tris in the pH range 7.0-9.0 or 0.01 M potassium phosphate in the pH range 6.0-6.5 and reacted with HRP. The best ratio of signal to reagent background resulted from reagents with a pH in the range 7 . 5-8 . 5 .
• Example 17 Effect of Buffer Salt.
• Detection reagent solutions according to the composition of example 14 were prepared with substitution of various buffer solutions and reacted with HRP. Useful levels of light intensity compared to reagent background were obtained with reagents prepared from tris hydrochloride, tris acetate, tris malate, potassium phosphate, diglycine-sodium hydroxide and tricine buffers.
• Example 18 Effect of Enhancers. Detection reagent solutions according to the composition of example 14 were prepared with substitution of various phenolic enhancers and reacted with HRP. Useful levels of light intensity compared to reagent background were obtained with reagents incorporating p-iodophenol, p-hydroxycinnamic acid and p-phenylphenol. Example 19. Effect of Peroxide. Detection reagent solutions according to the composition of example 14 were prepared with substitution of various peroxides and reacted with HRP. Useful levels of light intensity compared to reagent background were obtained with reagents incorporating hydrogen peroxide, sodium perborate and urea peroxide.
• Example 20 Effect of Surfactant. Detection reagent solutions according to the composition of example 14 were prepared with substitution of various surfactants and reacted with HRP. Useful levels of light intensity compared to reagent background were obtained with reagents incorporating TWEEN( 20 (Aldrich, Milwaukee, WI), TRITON( X-405 (Aldrich), BRIJ 35 (Aldrich), sodium dodecyl sulfate, cetyltrimethylammonium bromide, ⁇ -cyclodextrin and dextran sulfate.
• Example 21 Improved Chemiluminescent Detection of Proteins by Western Blot
• the proteins in the gel were electroeluted for 25 min at 4°C at a 100 V constant voltage.
• the membrane was then placed in 50 mM Tris-HCl buffered saline at pH 7.4 (TBS) at 4° C overnight. After this period the membrane was washed with TBS for 15 min.
• the membrane was treated with 0.05% TWEEN-20 in 50mM Tris-HCl buffered saline at pH 7.4 (T-TBS) containing 1% non-fat powdered milk (NFM) for one hour at room temperature. This blocked membrane was incubated for 75 minutes at room temperature with primary antibody (1:1500 dilution of goat anti-human transferrin IgG fraction) using T-TBS containing 1% NFM.
• T-TBS Tris-HCl buffered saline at pH 7.4
• NFM non-fat powdered milk
• the membrane was then rinsed and washed three times for five min each with T-TBS at room temperature.
• the washed membrane was incubated for one hour at room temperature with secondary antibody (1:50,000 dilution of rabbit anti-goat IgG peroxidase conjugate) using T-TBS containing 1% NFM.
• the membrane was rinsed and washed four times for ten minutes each with T-TBS followed by a five min wash with TBS.
• Reagent A was a commercial reagent containing luminol (Amersham ECL).
• Reagent B contained the acridan 4'-hydroxyphenyl 10-methylacridan-9-carboxylate previously disclosed in applicants' co-pending application Serial No. 08/061,810.
• Reagent C contained acridan 5e. After an incubation period of 15 min, the X-ray film was exposed to the membrane for varying periods of time and developed.
• the composition of detection reagent solution containing the acridan compounds was: Tris buffer, pH 8.8 0.1 M
• Figure 9 is a digitally scanned image of the X-ray film record of an experiment using a 14 min incubation and 15 s exposure.
• Example 22 Chemiluminescent Detection of Proteins by Western Blot using Nitrocellulose Membrane.
• a Western blot analysis of human transferrin was conducted by the method of example 21 with blotting of protein onto nitrocellulose in place of PVDF.
• the transferrin standards utilized were clearly visible down to 20 pg/slot over the background after a one min exposure to Kodak X-OMAT AR X-ray film. It was possible to make several exposures of the membrane over a period of 12 hours as the membrane continued to emit light.
• Figure 10 is a digitally scanned image of the X-ray film record of an experiment using a 15 min incubation and 1 min exposure.
• TSH Enzyme Immunoassay A TSH assay was performed using the components of a COBAS Core enzyme immunoassay kit for TSH from Roche (Basel, Switzerland) and a detection reagent of the present invention.
• the detection reagent comprised:
• Example 24 Human Growth Hormone Enzyme Immunoassay An hGH assay was performed using the components of a sandwich enzyme immunoassay kit for hGH from Sorin Biomedica (Vercelli, Italy) and a detection reagent of the present invention.
• the detection reagent comprised: Tris buffer, pH 8. 0 0.01 M
• the assay was repeated with a modification in which the secondary antibody-HRP conjugate supplied in the kit was diluted 10-fold with a dilution buffer supplied by the manufacturer.
• the assay resulted in a linear calibration curve over three orders of magnitude with a demonstrated detection limit of 0.05 ng/mL of hGH.
• Excellent linearity and identical analytical sensitivity resulted when light intensity was measured at 2.5, 5, 7.5 or 10 min ( Figure 13).
• Calibration data supplied by the manufacturer for the colorimetric method results in a nonlinear curve covering two orders of magnitude and requires a 30 min detection time.
• the calculated analytical sensitivity (signal > 2 standard deviations of blank) of the manufacturer's assay is 0.05 ng/mL.
• Example 25 Chemiluminescent Detection of Southern Blots.
• Mouse genomic DNA (Clontech Laboratories. Inc., Palo Alto, CA) was cleaved to completion with restriction endonuclease EcoR1 (Boehringer-Mannheim) at a concentration of 50 ⁇ g/mL.
• the restricted DNA was purified by extraction once with phenol/chloroform, once with chloroform and was precipitated with ethanol.
• the purified DNA was divided into two portions containing 30 and 15 ⁇ g of DNA, respectively and was separated by 0.77% agarose gel electrophoresis.
• the electrophoresis buffer was 40 mM Tris-acetate and 2 mM EDTA (pH 8.0). After electrophoresis the gel was rinsed with H 2 O and then soaked in 0.25 N HCl for 12 min with gentle agitation.
• MAGNAGRAPH NYLON (Micron Separations Inc., Westboro, MA) was soaked sequentially in water and 10X SSC (20X SSC is 3 M NaCl, 0.3 M sodium citrate, pH 7.0) for 2 and 10 min, respectively.
• the gel was rinsed with water and then treated with 0.5 M NaOH/1.5 M NaCl twice for 15 and 30 minutes, respectively.
• the gel was rinsed with water and then treated with 1 M Tris-HCl (pH 7.5)/1.5 M NaCl three times for 15 min each.
• the DNA in the gel was transferred onto the membrane by capillary blotting overnight using 10X SSC. The blots were air-dried for 30 min followed by baking at 80°C for 2 hours.
• the membranes were prehybridized in hybridization buffer (Amersham # RPN.3000) containing
• the membranes were rinsed with water and placed on 3MM blotting paper for one minute to remove excess solution, then transferred to a clean container followed by the addition of the detection reagent of Example 21. After a one minute incubation, excess solution was drained off and the blots were placed between sheets of transparency film followed by exposure to Kodak X-OMAT XAR 5 film.
• the reagent of the present invention can be used to detect a single copy gene in mouse genomic DNA as shown in Figure 14A.
• the target restriction fragment is 14 kb providing 70 pg (7 ⁇ 10 -17 moles) of target DNA in the 15 ⁇ g leading tracks.
• the single copy gene was clearly visible in both tracks of the blot using the detection reagent of the present invention ( Figure 14A) while the luminol reagent did not permit the bands to be detected ( Figure 14B). It is intended that the foregoing description be only illustrative of the present invention and that the present invention be limited only by the appended claims.

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